Neurosteroids Allosterically Modulate Binding of the Anesthetic Etomidate to γ-Aminobutyric Acid Type A Receptors

Photoaffinity labeling of gamma-aminobutyric acid type A (GABA(A))-receptors (GABA(A)R) with an etomidate analog and mutational analyses of direct activation of GABA(A)R by neurosteroids have each led to the proposal that these structurally distinct general anesthetics bind to sites in GABA(A)Rs in the transmembrane domain at the interface between the beta and alpha subunits. We tested whether the two ligand binding sites might overlap by examining whether neuroactive steroids inhibited etomidate analog photolabeling. We previously identified (Li, G. D., Chiara, D. C., Sawyer, G. W., Husain, S. S., Olsen, R. W., and Cohen, J. B. (2006) J. Neurosci. 26, 11599 - 11605) azietomidate photolabeling of GABA(A)R alpha 1Met-236 and beta Met-286 (in alpha M1 and beta M3). Positioning these two photolabeled amino acids in a single type of binding site at the interface of beta and alpha subunits (two copies per pentamer) is consistent with a GABA(A)R homology model based upon the structure of the nicotinic acetylcholine receptor and with recent alpha M1 to beta M3 cross-linking data. Biologically active neurosteroids enhance rather than inhibit azietomidate photolabeling, as assayed at the level of GABA(A)R subunits on analytical SDS-PAGE, and protein microsequencing establishes that the GABA(A)R-modulating neurosteroids do not inhibit photolabeling of GABA(A)R alpha 1Met-236 or beta Met-286 but enhance labeling of alpha 1Met-236. Thus modulatory steroids do not bind at the same site as etomidate, and neither of the amino acids identified as neurosteroid activation determinants (Hosie, A. M., Wilkins, M. E., da Silva, H. M., and Smart, T. G. (2006) Nature 444, 486 - 489) are located at the subunit interface defined by our etomidate site model.